CN107419021A - A kind of authentication method of wheat foreign gene insertion point - Google Patents
A kind of authentication method of wheat foreign gene insertion point Download PDFInfo
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- CN107419021A CN107419021A CN201710698017.2A CN201710698017A CN107419021A CN 107419021 A CN107419021 A CN 107419021A CN 201710698017 A CN201710698017 A CN 201710698017A CN 107419021 A CN107419021 A CN 107419021A
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- wheat
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- foreign gene
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
Abstract
The present invention relates to a kind of authentication method of wheat foreign gene insertion point, belong to genetically modified crops identification technology field.Authentication method provided by the invention comprises the following steps：1) Wheat volatiles are extracted；2) 3 nested special primer SP1, SP2 and SP3 are designed；3) specific primer SP1, SP2 and the SP3 obtained with step 2) is combined with chromosome walking kit carries out hot asymmetric interlaced PCR reactions, obtains amplified fragments；4) amplified fragments that step 3) obtains are sequenced into federation's Wheat volatiles data with international Wheat volatiles to be compared, draw insertion point of the foreign gene in wheat.Authentication method false positive probability of occurrence provided by the invention is low, and the insertion position of foreign gene can be precisely located, and for the later stage, the research of transfer-gen plant function has far-reaching significance, and educates new wheat germplasm resource for wound and lays a good foundation.
Description
Technical field
The present invention relates to genetically modified crops identification technology field, and in particular to a kind of mirror of wheat foreign gene insertion point
Determine method.
Background technology
Wheat is one of most important cereal crops in the world, and abundant protein and energy are provided for population in the world,
And the second largest cereal crops in China.With the increase of population, the following whole world will become to the demand of wheat in what is increased substantially
Gesture.Because the limitation of gene, the growth of wheat yield is conditional, it is impossible to by traditional cultivation technique come meet demand.
Transgenic technology is likely to become the important means for alleviating Food Security.For meet demand, it is necessary to pass through genetic engineering
Means cultivate genetically modified plants.Transgenic technology is a kind of flourishing means of development, can help to turn out with ideal character
Plant, there is great potential.Due to target gene inserts in transgenic technology randomness and insertion point gene expression
Blindness, the security of genetically modified crops cause the extensive concern of the public；And the cereal crop genetic transformation efficiency such as wheat
It is extremely low, and it is most important to find a kind of method of effective discriminating transgenic wheat.
The content of the invention
It is an object of the invention to provide a kind of authentication method of wheat foreign gene insertion point.Mirror provided by the invention
It is low to determine method false positive probability of occurrence, and the insertion position of foreign gene can be precisely located, method pair provided by the invention
In the later stage, the research of transfer-gen plant function has far-reaching significance, and educates new wheat germplasm resource for wound and lays a good foundation.
The invention provides a kind of authentication method of wheat foreign gene insertion point, comprise the following steps：
1) using the wheat after Agrobacterium-mediated transformation as material extraction Wheat volatiles；
2) principle according to Genome-walking, it is embedding based on the T-DNA sequences of step 1) conversion carrier, design 3
Special primer SP1, SP2 and SP3 of set；
3) specific primer SP1, SP2 and the SP3 obtained with step 2) is combined progress heat with chromosome walking kit not
Symmetrically staggeredly PCR reactions, obtain amplified fragments；
4) amplified fragments for obtaining step 3) and the Wheat volatiles data progress of international Wheat volatiles sequencing federation
Compare, draw insertion point of the foreign gene in wheat.
Preferably, the extraction of genome is carried out using wheat leaf blade.
Preferably, the chromosome walking kit is the chromosome walking kit that precious biotech firm sells；
The chromosome walking kit includes following components：Degenerate primer AP1, AP2, AP3 and AP4, contrast template,
TaKaRa LATaq、10×LAPCR Buffer II(Mg2+plus)、dNTP Mixture、6×Loading Buffer。
Preferably, the Tm values of 3 specific primers independently are 50~60 DEG C in the step 2).
Preferably, the conversion is pDTAAS with carrier.
Preferably, when conversion is pDTAAS with carrier, the sequence for the primer SP1 that the step 2) design obtains is such as
Shown in SEQ ID NO.1, SP2 sequence is as shown in SEQ ID NO.2, and SP3 sequence is as shown in SEQ ID NO.3.
Preferably, also include before carrying out comparing in the step 4)：It will be carried in the amplified fragments with conversion
The complementary gene segment of body removes.
The invention provides a kind of authentication method of wheat foreign gene insertion point.Using chromosome walking technology, together
When be combined with bioinformatic analysis, the insertion position of foreign gene can be precisely located in Wheat volatiles sequencing sequence
Put, insertion point be accurate to some site of some chromosome, filter out foreign gene insertion wheat cdna between or centromere
The transgenic line in region, interference of the foreign gene to Wheat volatiles can be prevented, and authentication method false positive of the present invention occurs
Probability is low, and for the later stage, the research of transfer-gen plant function has far-reaching significance, and educates new wheat germplasm resource for wound and establishes
Basis.
Brief description of the drawings
Fig. 1 is pDTAAS expression vectors collection of illustrative plates and special primer design attitude in the present invention；
Fig. 2 is that the combination chromosome walking kit that the embodiment of the present invention 1 provides carries out what hot asymmetric interlaced PCR was obtained
Extension increasing sequence band；
Fig. 3 is the foreign gene insertion point qualification result figure of sample 15 that the embodiment of the present invention 1 provides；
Fig. 4 is the foreign gene insertion point qualification result figure of sample 21 that the embodiment of the present invention 1 provides；
Fig. 5 is the foreign gene insertion point qualification result figure of sample 29 that the embodiment of the present invention 1 provides.
Embodiment
The invention provides a kind of authentication method of wheat foreign gene insertion point, comprise the following steps：
1) using the wheat after Agrobacterium-mediated transformation as material extraction Wheat volatiles；
2) principle according to Genome-walking, it is embedding based on the T-DNA sequences of step 1) conversion carrier, design 3
Special primer SP1, SP2 and SP3 of set；
3) specific primer SP1, SP2 and the SP3 obtained with step 2) is combined progress heat with chromosome walking kit not
Symmetrically staggeredly PCR reactions, obtain amplified fragments；
4) amplified fragments for obtaining step 3) and the Wheat volatiles data progress of international Wheat volatiles sequencing federation
Compare, draw insertion point of the foreign gene in wheat.
The present invention is using the wheat after Agrobacterium-mediated transformation as material extraction Wheat volatiles.In the present invention, it is preferred to
The extraction of genome is carried out using wheat leaf blade.The preferred wheat leaf blade for choosing seedling period of the invention, more preferably chooses light green
The wheat leaf blade unfolded is as extraction material.Specifically, during optimization experiment of the present invention clip 2cm length blade, put after shredding
Enter and frozen in 2mL centrifuge tubes, the temperature that freezes is preferably -80 DEG C.
The present invention does not have special restriction to the extracting method of the wheat leaf blade genome, using those skilled in the art
Well known plant genome DNA extracts kit is extracted, as Sangon Biotech (Shanghai) Co., Ltd. gives birth to
The Ezup pillar Plant Genome extracts kits of production.Specifically, in genome extraction process, the present invention is preferably by above-mentioned jelly
The wheat leaf blade grind into powder deposited, the extraction of genome is carried out according to the explanation of kit, obtained genome is preferably -20
DEG C preserved.
The present invention does not have special restriction to the operating method of the Agrobacterium-mediated transformation wheat, using this area skill
Agrobacterium_mediated method known to art personnel, expression vector such as is utilized, foreign gene is carried out by agrobacterium-mediated transformation
Conversion.The present invention does not have special restriction to the species of the conversion expression vector, using those skilled in the art
Well known agrobacterium-mediated transformation is often converted with expression vector.In the present invention, the conversion expression vector is preferred
For pDTAAS.
After obtaining Wheat volatiles, principle of the present invention according to Genome-walking, based on step 1) conversion carrier
T-DNA sequences, design 3 nested special primer SP1, SP2 and SP3.Present invention preferably uses chromosome walking kit
And the step of according to described in its specification, is operated.In the present invention, the chromosome walking kit is public for precious biology
The chromosome walking kit (Takara Genome Walking Kit, CodeNo.6108) of department, the chromosome walking examination
Agent box includes following components：Degenerate primer AP1, AP2, AP3 and AP4, contrast template, TaKaRa LATaq, 10 × LAPCR
Buffer II(Mg2+plus)、dNTP Mixture、6×Loading Buffer.Design principle of the present invention to special primer
It is preferred that with reference to the explanation in kit.Concrete operations refer to TaKaRa Genome Walking Kit specifications, herein without
Repeat.Specifically, in the present invention, when expression vector elects pDTAAS as, T-DNA sequence of the present invention preferably according to pDTAAS
Carry out the design of primer, in the specific embodiment of the invention, it is purpose gene to select SsNHX1, the insertion position of target gene with
And the design attitude of primer is as shown in Figure 1.According to T-DNA sequence areas (preferably equivalent at least 500bp) design three of known array
Specific primer：To need the foreign gene direction expanded, SP2 position should be designed in SP1 3 ' ends, SP3 positions design direction
In SP2 3 ' ends.The distance between each two primer does not have strict regulations, preferably 60~100bp.Fig. 1 shows primer
The binding site of SP1, SP2 and SP3 on carrier T-DNA regions, it is the sequence of insertion Plant Genome in theory from LB to RB.
In the present invention, the Tm values of 3 specific primers independently are 50~60 DEG C in the step 2).In the present invention
In, the primer that is obtained based on step 1) conversion with the LB borders of carrier to sequences Design between target gene be SP1, SP2 and
SP3；The expression direction for the foreign gene that design direction expands for needs, SP2 Position Design exist in SP1 3' ends, SP3 designs
SP2 3' ends.In the present invention, when conversion is pDTAAS with carrier, set based on step 1) conversion with the T-DNA sequences of carrier
Count primer SP1, SP2 and SP3, primer SP1 sequence as shown in SEQ ID NO.1, SP2 sequence as shown in SEQ ID NO.2,
SP3 sequence is as shown in SEQ ID NO.3.
After obtaining special primer, specific primer SP1, SP2 and SP3 and chromosome walking that the present invention is obtained with step 2)
Kit, which combines, carries out hot asymmetric interlaced PCR reactions, obtains amplified fragments.In the present invention, the chromosome walking reagent
Box is the chromosome walking kit that precious biotech firm sells, and concrete operation step refers to precious biological stain body walking kit
(TaKaRa Genome Walking Kit) specification.
After amplified fragments being obtained using the above method, amplified fragments and international wheat cdna that the present invention obtains step 3)
Group sequencing federation Wheat volatiles data are compared, and draw insertion point of the foreign gene in wheat.In the present invention,
Before comparison, preferably Hou Zaiyu worlds Wheat volatiles sequencing federation will be removed in amplified fragments with the complementary gene segment of carrier
Wheat volatiles are compared.The fragment that i.e. present invention amplification obtains is divided into two parts, a part and the partial order of expression vector
Row are complementary, and the sequence of another part and the gene at the Wheat volatiles of expression vector insertion is complementary, by by amplified fragments
In be compared again with Wheat volatiles after the gene segment shearing complementary with carrier, foreign gene can be realized in wheat cdna
The identification of specific insertion point in group.
A kind of authentication method of wheat foreign gene insertion point of the present invention is done with reference to specific embodiment
Further details of introduction, technical scheme include but is not limited to following examples.
Embodiment 1
First, the extraction of transgenic wheat genomic DNA
1) materials of transgenic wheat：After agrobacterium-mediated transformation obtains Transgenic plant of wheat, in seedling period, select tender
The green blade unfolded, using the scissors and tweezers after sterilizing, 2cm or so blade is cut, 2mL sterile centrifugation tubes are put into after shredding
In, -80 DEG C of Cord bloods.
2) extraction of genomic DNA：Using sterile grinding rod, the grind blade in liquid nitrogen, until being ground into what is turned white
Powder, using kit Sangon Biotech (Shanghai) Co., Ltd. Ezup pillar plant genome DNA extraction agent boxes
Model：B518261-0100, operating procedure extract wheat leaf blade genomic DNA as shown in illustrating kit.Obtain DNA solution,
Gently mix, it is of short duration to be collected by centrifugation to ttom of pipe, after taking 1 μ L extract solutions to measure concentration with micro ultraviolet specrophotometer, by DNA
Put -20 DEG C of preservations.
2nd, the identification of transgenic wheat
1. design primer
According to three specific primers of T-DNA sequences Designs of pDTAAS expression vectors, SP2 Position Design is SP1's
3' ends, SP3 is at SP2 3' ends.The zone of ignorance direction that design direction expands for needs.
| SEQ ID NO.1 | GENE-LBfor-SP1 | TGGTCATAGCTGTTTCCTGTGT |
| SEQ ID NO.2 | GENE-LBfor-SP2 | GTAAAGCCTGGGGTGCCTAA |
| SEQ ID NO.3 | GENE-LBfor-SP3 | TTCCAGTCGGGAAACCTGTC |
2. first round PCR is expanded
1) after transgenic wheat DNA solution is mixed, according to measurement concentration results, DNA is diluted to concentration 100ng/ μ L
Left and right, carry out first round amplification.
The Genome Walking Kit kits that experiment is provided using Takara companies.
Four kinds of relatively low degenerate primers of annealing temperature, i.e. AP1, AP2, AP3, AP4, this experimental design are provided in kit
Three in the same direction and higher specific primer SP1, SP2, the SP3 of annealing temperature.
2) 4 200 μ LPCR pipes are taken to enter performing PCR reaction.After reagent in kit is placed on ice into slowly thawing, gently
Mix, of short duration centrifugation.
PCR amplification system is：Total system 25 μ L, μ L of DNA profiling 0.5, the μ L of 10 × PCRbuffer 2.5 are reacted,
The μ of 4 μ L, SP10.5 μ L, APPrimers Template0.5 μ L, LATaq DNApolymerase of 2.5mmol/LdNTPs 0.25
L, ddH2O16.75μL.Wherein, degenerate primer added in each PCR pipe is different, is sequentially AP1, AP2, AP3, AP4.
PCR response procedures are：94 DEG C of 1min, 98 DEG C of 1min, the programs of 5 circulations are 94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C
2min, then 94 DEG C of 30s, 25 DEG C of 3min, 72 DEG C of 2min, the programs of following 15 circulations are 94 DEG C of 30s, 65 DEG C of 1min, 72
DEG C 2min, 94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C of 2min, 94 DEG C of 30s, 44 DEG C of 1min, 72 DEG C of 2min, last 72 DEG C of 10min.PCR
The template that product will expand as next round PCR.
3. the second wheel PCR amplifications
After first round amplified production is placed in into cooling on ice, gently mix, of short duration centrifugation.The system and the of second wheel amplification
One wheel is similar, but needs to use and expanded with first round identical degenerate primer, i.e., the suitable of AP primers is added in each PCR pipe
Sequence is AP1, AP2, AP3, AP4, and the specific primer used is SP2.The PCR liquid expanded in the first round using identical AP primers is made
For the template of the second wheel PCR reactions.
PCR amplification system is：10 × PCRbuffer 2.5 μ L, 2.5mmol/L dNTPs4 μ L, SP20.5 μ L, AP
Primers Template 0.5 μ L, LATaq DNApolymerase0.25 μ L, ddH2O16.75μL。
PCR response procedures are：15 circulation programs be：94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C of 2min, 94 DEG C of 30s, 65 DEG C
1min, 72 DEG C of 2min, 94 DEG C of 30s, 44 DEG C of 1min, 72 DEG C of 2min, last 72 DEG C of 10min.PCR primer will be used as third round PCR
The template of amplification.
4. third round PCR is expanded
After second wheel amplified production is placed in into cooling on ice, gently mix, of short duration centrifugation.Template as wheel PCR.For
Aspect electrophoresis detection and sequencing, third round amplification system double, and the response procedures of amplification are similar to the second wheel.This secondary response uses
Being expanded with the second wheel identical degenerate primer, i.e., the order that AP primers are added in each PCR pipe is AP1, AP2, AP3, AP4,
The specific primer used is SP3.The mould for using the PCR liquid of identical AP primers amplification to be reacted as third round PCR in second wheel
Plate.
PCR amplification system is：10 × PCRbuffer 5 μ L, 2.5mmol/L dNTPs8 μ L, SP31 μ L, AP Primers
μ L, LATaq DNApolymerase0.5 μ L, the ddH2O33.5 μ L of Template 1.
PCR response procedures are identical with the second wheel program：15 circulation programs be：94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C
2min, 94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C of 2min, 94 DEG C of 30s, 44 DEG C of 1min, 72 DEG C of 2min, last 72 DEG C of 10min.
5. electrophoresis detection
PCR liquid is gently mixed, of short duration centrifugation.10 μ L are taken, 26 × Buffer of μ L mixings is added and is used as electrophoresis Sample, make
Electrophoresis detection is carried out with the Ago-Gel that concentration is 1.5%.It will can detect the third round product of clear, bright band
PCR liquid is delivered to the prosperous company of Beijing AudioCodes and is sequenced, and primer uses SP3.
6. sequencing sequence removes the T-DNA flanking sequences after carrier sequence, federation is sequenced with international Wheat volatiles
(International wheat genome sequencing consortium) Wheat volatiles comparing.
7. all Transgenic plant of wheat are identified using the above method, reflected in three kinds of Transgenic plant of wheat
Counted after making three plants of positive plants, identify 254 plants of wheats using TAIL-PCR methods, Gel electrophoresis results show 46
There is clearly bright band in sample amplification, and sequencing result, which is shown, shares 9 plants of transgenic wheats, and foreign gene is successfully inserted into
Into its genome.
It is specifically described so that the sample in the transformation event that SsNHX1 is purpose gene is identified using this method as an example.
The fragment that extraction plant DNA is expanded to obtain detects under 1.2% agarose gel electrophoresis, third round PCR amplification purpose pieces
Sequencing is sent after section gel extraction, electrophoresis result is third round PCR amplification productions as shown in Fig. 2 the signified as sequencing fragment of arrow
Thing.Sequencing result is compared with carrier pDTAAS-SsNHX1 sequences using bioanalysis software geneious, compares knot
Fruit shows：15th, 21,29 samples sequencing acquired results can be consistent with carrier T-DNA left margins 200bp or so base sequence.Side
In frame for the genome portion sequence in sequencing sequence, compared unanimously with international Wheat volatiles sequencing sequence.Arrow is signified
As T-DNA insertion points on Wheat volatiles, further Blastn research and analyse result and shown, sample 15 is inserted into B groups
The 147705-148494 positions (Fig. 3) of wheat Article 6 chromosome long arm, same analysis method are drawn：Sample 21 is inserted into B
The 7134-7511 positions (Fig. 4) of group wheat Article 3 chromosome, sample 29 are inserted into the 7135- of B group wheat Article 3 chromosomes
7512 positions (Fig. 5).
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>TanJin Agricultural College
<120>A kind of authentication method of wheat foreign gene insertion point
<130> 2017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
tggtcatagc tgtttcctgt gt 22
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gtaaagcctg gggtgcctaa 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
ttccagtcgg gaaacctgtc 20
Claims (7)
1. a kind of authentication method of wheat foreign gene insertion point, comprises the following steps：
1) using the wheat after Agrobacterium-mediated transformation as material extraction Wheat volatiles；
2) principle according to Genome-walking, based on the T-DNA sequences of step 1) conversion carrier, design 3 it is nested
Special primer SP1, SP2 and SP3；
3) it is asymmetric that specific primer SP1, SP2 and the SP3 obtained with step 2) is combined progress heat with chromosome walking kit
Staggeredly PCR reacts, and obtains amplified fragments；
4) amplified fragments that step 3) obtains are sequenced into federation's Wheat volatiles data with international Wheat volatiles to be compared,
Draw insertion point of the foreign gene in wheat.
2. authentication method according to claim 1, it is characterised in that the extraction of genome is carried out using wheat leaf blade.
3. authentication method according to claim 1, it is characterised in that the chromosome walking kit is precious biotech firm
The chromosome walking kit of sale；
The chromosome walking kit includes following components：Degenerate primer AP1, AP2, AP3 and AP4, contrast template, TaKaRa
LA Taq、10×LAPCR Buffer II(Mg2+plus)、dNTP Mixture、6×Loading Buffer。
4. authentication method according to claim 1, it is characterised in that the Tm values of 3 specific primers in the step 2)
It independently is 50~60 DEG C.
5. authentication method according to claim 1, it is characterised in that the conversion is pDTAAS with carrier.
6. authentication method according to claim 5, it is characterised in that when conversion is pDTAAS with carrier, the step
2) sequence for the primer SP1 that design obtains is as shown in SEQ ID NO.1, and SP2 sequence is as shown in SEQ ID NO.2, SP3 sequence
Row are as shown in SEQ ID NO.3.
7. authentication method according to claim 1, it is characterised in that also wrapped before carrying out comparing in the step 4)
Include：It will be removed in the amplified fragments with conversion with the complementary gene segment of carrier.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110349624A (en) * | 2019-05-30 | 2019-10-18 | 山东省农业科学院玉米研究所 | The method of sam file f lag tag location T-DNA insertion point |
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| CN110349624A (en) * | 2019-05-30 | 2019-10-18 | 山东省农业科学院玉米研究所 | The method of sam file f lag tag location T-DNA insertion point |
| CN110349624B (en) * | 2019-05-30 | 2021-09-21 | 山东省农业科学院玉米研究所 | Method for positioning T-DNA insertion site by sam file flag tag |
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Application publication date: 20171201 |